Human nucleotidases are enzymes that terminate purinergic receptor-mediated responses, including many processes dependent upon extracellular ATP and ADP. The potential clinical utility of injecting soluble nucleotidases therapeutically to modulate these physiologically important processes has already been demonstrated. One specific therapeutic use of such soluble nucleotidases is the control of the pathophysiological blood clotting that causes heart attacks and strokes, via hydrolysis of ADP that triggers platelet activation and subsequent blood coagulation. Thus, one goal of the proposed research is to elucidate the structures of the soluble human nucleotidases and to use this information to understand their functions. A second goal is to use the structural information to design modified nucleotidases, having increased activity and specificity for nucleotides modulating physiological processes. Elucidating the structural determinants of substrate hydrolysis and specificity is crucial for control of important, purinergically controlled, processes. The two known, naturally occurring, soluble forms of human ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) will be expressed in bacteria and refolded to generate large quantities of enzymatically active, soluble eNTPDases. The expressed proteins will be purified, enzymatically characterized, and crystallized to determine their structures. Affinity labeling, site-directed mutagenesis, and computer modeling will be used to determine the roles played by individual amino acid residues in nucleotide hydrolysis, as well as to identify residues important for nucleotidase specificity. These results will be compared and contrasted with similar results obtained using another soluble human nucleotidase, newly discovered by our laboratory, which is related in sequence to a nucleotidase found in the saliva of bloodsucking insects, but unrelated in sequence to the eNTPDases. The study of this nucleotidase is important for two reasons. First, it represents a second potential therapeutic avenue for developing therapeutic soluble nucleotidases. Second, it will demonstrate how two different protein primary structures are used to do the same enzymatic task, aiding in the design of more efficacious therapeutic soluble nucleotidases.